@article{HenkelKlauderStatzetal.2021, author = {Henkel, Janin and Klauder, Julia and Statz, Meike and Wohlenberg, Anne-Sophie and Kuipers, Sonja and Vahrenbrink, Madita and P{\"u}schel, Gerhard Paul}, title = {Enhanced Palmitate-Induced Interleukin-8 Formation in Human Macrophages by Insulin or Prostaglandin E-2}, series = {Biomedicines}, volume = {9}, journal = {Biomedicines}, number = {5}, publisher = {MDPI}, address = {Basel}, issn = {2227-9059}, doi = {10.3390/biomedicines9050449}, pages = {10}, year = {2021}, abstract = {Macrophages in pathologically expanded dysfunctional white adipose tissue are exposed to a mix of potential modulators of inflammatory response, including fatty acids released from insulin-resistant adipocytes, increased levels of insulin produced to compensate insulin resistance, and prostaglandin E-2 (PGE(2)) released from activated macrophages. The current study addressed the question of how palmitate might interact with insulin or PGE(2) to induce the formation of the chemotactic pro-inflammatory cytokine interleukin-8 (IL-8). Human THP-1 cells were differentiated into macrophages. In these macrophages, palmitate induced IL-8 formation. Insulin enhanced the induction of IL-8 formation by palmitate as well as the palmitate-dependent stimulation of PGE(2) synthesis. PGE(2) in turn elicited IL-8 formation on its own and enhanced the induction of IL-8 release by palmitate, most likely by activating the EP4 receptor. Since IL-8 causes insulin resistance and fosters inflammation, the increase in palmitate-induced IL-8 formation that is caused by hyperinsulinemia and locally produced PGE(2) in chronically inflamed adipose tissue might favor disease progression in a vicious feed-forward cycle.}, language = {en} } @article{GehreFlechnerKammereretal.2020, author = {Gehre, Christian and Flechner, Marie and Kammerer, Sarah and K{\"u}pper, Jan-Heiner and Coleman, Charles Dominic and P{\"u}schel, Gerhard Paul and Uhlig, Katja and Duschl, Claus}, title = {Real time monitoring of oxygen uptake of hepatocytes in a microreactor using optical microsensors}, series = {Scientific reports}, volume = {10}, journal = {Scientific reports}, number = {1}, publisher = {Macmillan Publishers Limited, part of Springer Nature}, address = {[London]}, issn = {2045-2322}, doi = {10.1038/s41598-020-70785-6}, pages = {12}, year = {2020}, abstract = {Most in vitro test systems for the assessment of toxicity are based on endpoint measurements and cannot contribute much to the establishment of mechanistic models, which are crucially important for further progress in this field. Hence, in recent years, much effort has been put into the development of methods that generate kinetic data. Real time measurements of the metabolic activity of cells based on the use of oxygen sensitive microsensor beads have been shown to provide access to the mode of action of compounds in hepatocytes. However, for fully exploiting this approach a detailed knowledge of the microenvironment of the cells is required. In this work, we investigate the cellular behaviour of three types of hepatocytes, HepG2 cells, HepG2-3A4 cells and primary mouse hepatocytes, towards their exposure to acetaminophen when the availability of oxygen for the cell is systematically varied. We show that the relative emergence of two modes of action, one NAPQI dependent and the other one transient and NAPQI independent, scale with expression level of CYP3A4. The transient cellular response associated to mitochondrial respiration is used to characterise the influence of the initial oxygen concentration in the wells before exposure to acetaminophen on the cell behaviour. A simple model is presented to describe the behaviour of the cells in this scenario. It demonstrates the level of control over the role of oxygen supply in these experiments. This is crucial for establishing this approach into a reliable and powerful method for the assessment of toxicity.}, language = {en} } @article{SchenkeSchjeidePuescheletal.2020, author = {Schenke, Maren and Schjeide, Brit-Maren and P{\"u}schel, Gerhard Paul and Seeger, Bettina}, title = {Analysis of motor neurons differentiated from human induced pluripotent stem cells for the use in cell-based Botulinum neurotoxin activity assays}, series = {Toxins}, volume = {12}, journal = {Toxins}, number = {5}, publisher = {MDPI}, address = {Basel}, issn = {2072-6651}, doi = {10.3390/toxins12050276}, pages = {20}, year = {2020}, abstract = {Botulinum neurotoxins (BoNTs) are potent neurotoxins produced by bacteria, which inhibit neurotransmitter release, specifically in their physiological target known as motor neurons (MNs). For the potency assessment of BoNTs produced for treatment in traditional and aesthetic medicine, the mouse lethality assay is still used by the majority of manufacturers, which is ethically questionable in terms of the 3Rs principle. In this study, MNs were differentiated from human induced pluripotent stem cells based on three published protocols. The resulting cell populations were analyzed for their MN yield and their suitability for the potency assessment of BoNTs. MNs produce specific gangliosides and synaptic proteins, which are bound by BoNTs in order to be taken up by receptor-mediated endocytosis, which is followed by cleavage of specific soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) proteins required for neurotransmitter release. The presence of receptors and substrates for all BoNT serotypes was demonstrated in MNs generated in vitro. In particular, the MN differentiation protocol based on Du et al. yielded high numbers of MNs in a short amount of time with high expression of BoNT receptors and targets. The resulting cells are more sensitive to BoNT/A1 than the commonly used neuroblastoma cell line SiMa. MNs are, therefore, an ideal tool for being combined with already established detection methods.}, language = {en} } @article{SchaeferKakularamReischetal.2022, author = {Sch{\"a}fer, Marj{\"a}nn Helena and Kakularam, Kumar Reddy and Reisch, Florian and Rothe, Michael and Stehling, Sabine and Heydeck, Dagmar and P{\"u}schel, Gerhard Paul and Kuhn, Hartmut}, title = {Male Knock-in Mice Expressing an Arachidonic Acid Lipoxygenase 15B (Alox15B) with Humanized Reaction Specificity Are Prematurely Growth Arrested When Aging}, series = {Biomedicines}, volume = {10}, journal = {Biomedicines}, edition = {6}, publisher = {MDPI}, address = {Basel, Schweiz}, issn = {2227-9059}, doi = {10.3390/biomedicines10061379}, pages = {1 -- 22}, year = {2022}, abstract = {Mammalian arachidonic acid lipoxygenases (ALOXs) have been implicated in cell differentiation and in the pathogenesis of inflammation. The mouse genome involves seven functional Alox genes and the encoded enzymes share a high degree of amino acid conservation with their human orthologs. There are, however, functional differences between mouse and human ALOX orthologs. Human ALOX15B oxygenates arachidonic acid exclusively to its 15-hydroperoxy derivative (15S-HpETE), whereas 8S-HpETE is dominantly formed by mouse Alox15b. The structural basis for this functional difference has been explored and in vitro mutagenesis humanized the reaction specificity of the mouse enzyme. To explore whether this mutagenesis strategy may also humanize the reaction specificity of mouse Alox15b in vivo, we created Alox15b knock-in mice expressing the arachidonic acid 15-lipoxygenating Tyr603Asp+His604Val double mutant instead of the 8-lipoxygenating wildtype enzyme. These mice are fertile, display slightly modified plasma oxylipidomes and develop normally up to an age of 24 weeks. At later developmental stages, male Alox15b-KI mice gain significantly less body weight than outbred wildtype controls, but this effect was not observed for female individuals. To explore the possible reasons for the observed gender-specific growth arrest, we determined the basic hematological parameters and found that aged male Alox15b-KI mice exhibited significantly attenuated red blood cell parameters (erythrocyte counts, hematocrit, hemoglobin). Here again, these differences were not observed in female individuals. These data suggest that humanization of the reaction specificity of mouse Alox15b impairs the functionality of the hematopoietic system in males, which is paralleled by a premature growth arrest.}, language = {en} } @article{HenkelColemanSchraplauetal.2017, author = {Henkel, Janin and Coleman, Charles Dominic and Schraplau, Anne and J{\"o}hrens, Korinna and Weber, Daniela and Castro, Jose Pedro and Hugo, Martin and Schulz, Tim Julius and Kr{\"a}mer, Stephanie and Sch{\"u}rmann, Annette and P{\"u}schel, Gerhard Paul}, title = {Induction of Steatohepatitis (NASH) with Insulin Resistance in Wild-type B6 Mice by a Western-type Diet Containing Soybean Oil and Cholesterol}, series = {Molecular medicine}, volume = {23}, journal = {Molecular medicine}, publisher = {Feinstein Inst. for Medical Research}, address = {Manhasset}, issn = {1076-1551}, doi = {10.2119/molmed.2016.00203}, pages = {70 -- 82}, year = {2017}, abstract = {Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are hepatic manifestations of the metabolic syndrome. Many currently used animal models of NAFLD/NASH lack clinical features of either NASH or metabolic syndrome such as hepatic inflammation and fibrosis (e.g., high-fat diets) or overweight and insulin resistance (e.g., methionine-choline-deficient diets), or they are based on monogenetic defects (e.g., ob/ob mice). In the current study, a Western-type diet containing soybean oil with high n-6-PUFA and 0.75\% cholesterol (SOD + Cho) induced steatosis, inflammation and fibrosis accompanied by hepatic lipid peroxidation and oxidative stress in livers of C57BL/6-mice, which in addition showed increased weight gain and insulin resistance, thus displaying a phenotype closely resembling all clinical features of NASH in patients with metabolic syndrome. In striking contrast, a soybean oil-containing Western-type diet without cholesterol (SOD) induced only mild steatosis but not hepatic inflammation, fibrosis, weight gain or insulin resistance. Another high-fat diet, mainly consisting of lard and supplemented with fructose in drinking water (LAD + Fru), resulted in more prominent weight gain, insulin resistance and hepatic steatosis than SOD + Cho, but livers were devoid of inflammation and fibrosis. Although both LAD + Fru-and SOD + Cho-fed animals had high plasma cholesterol, liver cholesterol was elevated only in SOD + Cho animals. Cholesterol induced expression of chemotactic and inflammatory cytokines in cultured Kupffer cells and rendered hepatocytes more susceptible to apoptosis. In summary, dietary cholesterol in the SOD + Cho diet may trigger hepatic inflammation and fibrosis. SOD + Cho-fed animals may be a useful disease model displaying many clinical features of patients with the metabolic syndrome and NASH.}, language = {en} } @article{NeuschaeferRubePatheNeuschaeferRubePueschel2022, author = {Neusch{\"a}fer-Rube, Frank and Pathe-Neusch{\"a}fer-Rube, Andrea and P{\"u}schel, Gerhard Paul}, title = {Discrimination of the activity of low-affinity wild-type and high-affinity mutant recombinant BoNT/B by a SIMA cell-based reporter release assay}, series = {Toxins}, volume = {14}, journal = {Toxins}, edition = {1}, publisher = {MDPI}, address = {Basel, Schweiz}, issn = {2072-6651}, doi = {10.3390/toxins14010065}, pages = {1 -- 11}, year = {2022}, abstract = {Botulinum neurotoxin (BoNT) is used for the treatment of a number of ailments. The activity of the toxin that is isolated from bacterial cultures is frequently tested in the mouse lethality assay. Apart from the ethical concerns inherent to this assay, species-specific differences in the affinity for different BoNT serotypes give rise to activity results that differ from the activity in humans. Thus, BoNT/B is more active in mice than in humans. The current study shows that the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-Gluc) was inhibited by clostridial and recombinant BoNT/A to the same extent, whereas both clostridial and recombinant BoNT/B inhibited the release to a lesser extent and only at much higher concentrations, reflecting the low activity of BoNT/B in humans. By contrast, the genetically modified BoNT/B-MY, which has increased affinity for human synaptotagmin, and the BoNT/B protein receptor inhibited luciferase release effectively and with an EC50 comparable to recombinant BoNT/A. This was due to an enhanced uptake into the reporter cells of BoNT/B-MY in comparison to the recombinant wild-type toxin. Thus, the SIMA-hPOMC1-26-Gluc cell assay is a versatile tool to determine the activity of different BoNT serotypes providing human-relevant dose-response data.}, language = {en} } @article{vonLoeffelholzLieskeNeuschaeferRubeetal.2017, author = {von Loeffelholz, Christian and Lieske, Stefanie and Neuschaefer-Rube, Frank and Willmes, Diana M. and Raschzok, Nathanael and Sauer, Igor M. and K{\"o}nig, J{\"o}rg and Fromm, Martin F. and Horn, Paul and Chatzigeorgiou, Antonios and Pathe-Neuschaefer-Rube, Andrea and Jordan, Jens and Pfeiffer, Andreas F. H. and Mingrone, Geltrude and Bornstein, Stefan R. and Stroehle, Peter and Harms, Christoph and Wunderlich, F. Thomas and Helfand, Stephen L. and Bernier, Michel and de Cabo, Rafael and Shulman, Gerald I. and Chavakis, Triantafyllos and P{\"u}schel, Gerhard Paul and Birkenfeld, Andreas L.}, title = {The human longevity gene homolog INDY and interleukin-6 interact in hepatic lipid metabolism}, series = {Hepatology}, volume = {66}, journal = {Hepatology}, number = {2}, publisher = {Wiley}, address = {Hoboken}, issn = {0270-9139}, doi = {10.1002/hep.29089}, pages = {616 -- 630}, year = {2017}, abstract = {Reduced expression of the Indy ("I am Not Dead, Yet") gene in lower organisms promotes longevity in a manner akin to caloric restriction. Deletion of the mammalian homolog of Indy (mIndy, Slc13a5) encoding for a plasma membrane-associated citrate transporter expressed highly in the liver, protects mice from high-fat diet-induced and aging-induced obesity and hepatic fat accumulation through a mechanism resembling caloric restriction. We studied a possible role of mIndy in human hepatic fat metabolism. In obese, insulin-resistant patients with nonalcoholic fatty liver disease, hepatic mIndy expression was increased and mIndy expression was also independently associated with hepatic steatosis. In nonhuman primates, a 2-year high-fat, high-sucrose diet increased hepatic mIndy expression. Liver microarray analysis showed that high mIndy expression was associated with pathways involved in hepatic lipid metabolism and immunological processes. Interleukin-6 (IL-6) was identified as a regulator of mIndy by binding to its cognate receptor. Studies in human primary hepatocytes confirmed that IL-6 markedly induced mIndy transcription through the IL-6 receptor and activation of the transcription factor signal transducer and activator of transcription 3, and a putative start site of the human mIndy promoter was determined. Activation of the IL-6-signal transducer and activator of transcription 3 pathway stimulated mIndy expression, enhanced cytoplasmic citrate influx, and augmented hepatic lipogenesis in vivo. In contrast, deletion of mIndy completely prevented the stimulating effect of IL-6 on citrate uptake and reduced hepatic lipogenesis. These data show that mIndy is increased in liver of obese humans and nonhuman primates with NALFD. Moreover, our data identify mIndy as a target gene of IL-6 and determine novel functions of IL-6 through mINDY. Conclusion: Targeting human mINDY may have therapeutic potential in obese patients with nonalcoholic fatty liver disease. German Clinical Trials Register: DRKS00005450.}, language = {en} } @article{KnebelNeebZahnetal.2018, author = {Knebel, Constanze and Neeb, Jannika and Zahn, Elisabeth and Schmidt, Flavia and Carazo, Alejandro and Holas, Ondej and Pavek, Petr and P{\"u}schel, Gerhard Paul and Zanger, Ulrich M. and S{\"u}ssmuth, Roderich and Lampen, Alfonso and Marx-Stoelting, Philip and Braeuning, Albert}, title = {Unexpected Effects of Propiconazole, Tebuconazole, and Their Mixture on the Receptors CAR and PXR in Human Liver Cells}, series = {Toxicological sciences}, volume = {163}, journal = {Toxicological sciences}, number = {1}, publisher = {Oxford Univ. Press}, address = {Oxford}, issn = {1096-6080}, doi = {10.1093/toxsci/kfy026}, pages = {170 -- 181}, year = {2018}, abstract = {Analyzing mixture toxicity requires an in-depth understanding of the mechanisms of action of its individual components. Substances with the same target organ, same toxic effect and same mode of action (MoA) are believed to cause additive effects, whereas substances with different MoAs are assumed to act independently. Here, we tested 2 triazole fungicides, propiconazole, and tebuconazole (Te), for individual and combined effects on liver toxicity-related endpoints. Both triazoles are proposed to belong to the same cumulative assessment group and are therefore thought to display similar and additive behavior. Our data show that Te is an antagonist of the constitutive androstane receptor (CAR) in rats and humans, while propiconazole is an agonist of this receptor. Both substances activate the pregnane X-receptor (PXR) and further induce mRNA expression of CYP3A4. CYP3A4 enzyme activity, however, is inhibited by propiconazole. For common targets of PXR and CAR, the activation of PXR by Te overrides CAR inhibition. In summary, propiconazole and Te affect different hepatotoxicity-relevant cellular targets and, depending on the individual endpoint analyzed, act via similar or dissimilar mechanisms. The use of molecular data based on research in human cell systems extends the picture to refine cumulative assessment group grouping and substantially contributes to the understanding of mixture effects of chemicals in biological systems.}, language = {en} } @article{HenkelColemanSchraplauetal.2018, author = {Henkel, Janin and Coleman, Charles Dominic and Schraplau, Anne and Joehrens, Korinna and Weiss, Thomas Siegfried and Jonas, Wenke and Sch{\"u}rmann, Annette and P{\"u}schel, Gerhard Paul}, title = {Augmented liver inflammation in a microsomal prostaglandin E synthase 1 (mPGES-1)-deficient diet-induced mouse NASH model}, series = {Scientific reports}, volume = {8}, journal = {Scientific reports}, publisher = {Nature Publ. Group}, address = {London}, issn = {2045-2322}, doi = {10.1038/s41598-018-34633-y}, pages = {11}, year = {2018}, abstract = {In a subset of patients, non-alcoholic fatty liver disease (NAFLD) is complicated by cell death and inflammation resulting in non-alcoholic steatohepatitis (NASH), which may progress to fibrosis and subsequent organ failure. Apart from cytokines, prostaglandins, in particular prostaglandin E-2 (PGE(2)), play a pivotal role during inflammatory processes. Expression of the key enzymes of PGE(2) synthesis, cyclooxygenase 2 and microsomal PGE synthase 1 (mPGES-1), was increased in human NASH livers in comparison to controls and correlated with the NASH activity score. Both enzymes were also induced in NASH-diet-fed wild-type mice, resulting in an increase in hepatic PGE(2) concentration that was completely abrogated in mPGES-1-deficient mice. PGE(2) is known to inhibit TNF-alpha synthesis in macrophages. A strong infiltration of monocyte-derived macrophages was observed in NASH-diet-fed mice, which was accompanied with an increase in hepatic TNF-alpha expression. Due to the impaired PGE(2) production, TNF-alpha expression increased much more in livers of mPGES-1-deficient mice or in the peritoneal macrophages of these mice. The increased levels of TNF-alpha resulted in an enhanced IL-1 beta production, primarily in hepatocytes, and augmented hepatocyte apoptosis. In conclusion, attenuation of PGE(2) production by mPGES-1 ablation enhanced the TNF-alpha-triggered inflammatory response and hepatocyte apoptosis in diet-induced NASH.}, language = {en} } @article{NeuschaeferRubePatheNeuschaeferRubeHippenstieletal.2018, author = {Neusch{\"a}fer-Rube, Frank and Pathe-Neusch{\"a}fer-Rube, Andrea and Hippenstiel, Stefan and P{\"u}schel, Gerhard Paul}, title = {PGE(2) enhanced TNF alpha-mediated IL-8 induction in monocytic cell lines and PBMC}, series = {Cytokine}, volume = {113}, journal = {Cytokine}, publisher = {Elsevier}, address = {London}, issn = {1043-4666}, doi = {10.1016/j.cyto.2018.06.020}, pages = {105 -- 116}, year = {2018}, abstract = {Background \& purpose: Recent studies suggested a role of prostaglandin E-2 (PGE(2)) in the expression of the chemokine IL-8 by monocytes. The function of EP4 receptor for TNF alpha-induced IL-8 expression was studied in monocytic cell lines. Experimental approach: IL-8 mRNA and protein induction as well as IL-8 promoter activity and transcription factor activation were assessed in monocytic cell lines, primary blood mononuclear cells (PBMC) and transgenic HEK293 cells expressing the EP4 receptor. Key results: In monocytic cell lines THP-1, MonoMac and U937 PGE(2) had only a marginal impact on IL-8 induction but strongly enhanced TNFa-induced IL-8 mRNA and protein synthesis. Similarly, in PBMC IL-8 mRNA induction was larger by simultaneous stimulation with TNF alpha and PGE(2) than by either stimulus alone. The EP4 receptor subtype was the most abundant EP receptor in all three cell lines and in PBMC. Stimulation of THP-1 cells with an EP4 specific agonist enhanced TNF alpha-induced IL-8 mRNA and protein formation to the same extent as PGE(2). In HEK293 cells expressing EP4, but not in wild type HEK293 cells lacking EP4, PGE(2) enhanced TNFainduced IL-8 protein and mRNA synthesis. In THP-1 cells, the enhancement of TNF alpha-mediated IL-8 mRNA induction by PGE(2) was mimicked by a PICA-activator. Furthermore in these cells PGE(2) induced expression of transcription factor C/EBPS, enhanced NF-KB activation by TNFa and inhibited TNF alpha-mediated AP-1 activation. PGE(2) and TNF alpha synergistically activated transcription factor CREB, induced C/EBPS expression and enhanced the activity of an IL-8 promoter fragment containing-223 bp upstream of the transcription start site. Conclusions and implications: These findings suggest that a combined stimulation of TNF alpha and PGE(2)/EP4 signal chains in monocytic cells leads to maximal IL-8 promoter activity, as well as IL-8 mRNA and protein induction, by activating the PICA/CREB/C/EB1313 as well as NF-kappa B signal chains.}, language = {en} }